Endocytosis by clathrin coated vesicles is a fundamental mechanism used by all eukaryotic cells to maintain their plasma membrane, communicate with the outside world, and internalize nutrients. Our long-term goal is to understand the molecular basis for the formation and regulation of clathrin vesicles, important contributors to endocytosis. Recently it has become clear that the actin cytoskeleton interacts with the clathrin machinery, and contributes filaments that contribute to the formation of coated vesicles. A critical gap in the field is the lack of a molecular mechanism that could explain how clathrin assembly and actin polymerization are coordinated temporally and spatially in living cells to efficiently form a coated vesicle. The objective of this proposal is to define the mechanism by which the proteins epsin, Hip1r and the clathrin light chain interact to promote the formation of functional clathrin coated vesicles coupled to a focused array of actin filaments in living cells. Using Dictyostelium cells as a model system, three specific aims are proposed: (1) To define how Hip1r regulates coupling of actin filaments to clathrin Our initial results show that Hip1r contributes to the timing and the morphology of a tightly focused band of actin filaments with coated pits. Two working models for these Hip1r activities will be tested. Informative point mutants in distinct domains of Hip1r will be evaluated to distinguish between the possibility that Hip1r acts as a regulated tether between clathrin and actin, and the possibility that Hip1r serves as a scaffold to recruit actin-regulatory proteins to clathrin lattices. (2)To determine how phosphorylation and epsin regulate Hip1r function Initial studies suggest that epsin contributes to a pathway that regulates Hip1r phosphorylation and activity. Mass spectrometry will verify a candidate Hip1r phosphorylation site. Experiments using phosphosilent and phosphomimic versions of Hip1r will directly test (a) whether phosphorylation controls Hip1r activity, and (b) how phosphorylation may regulate Hip1r activity. The possibility that epsin controls Hip1r through phosphorylation will be tested and binding partners for epsin that serves as intermediates in this pathway will be identified. A candidate kinase for Hip1r will be tested (3) To define the contribution of the clathrin light chain contributes to clathrin function Using quick-freeze deep etch microscopy, the contribution of the clathrin light chain (CLC) to the trimerization of heavy chain and to the architecture of coated pits assembled in living cells will be evaluated. Total interference microscopy will evaluate the contribution of the CLC to the dynamic assembly of clathrin and actin on the plasma membrane. A collection of five defined truncations of CLC will be examined for contributions to specific aspects of coated pit formation, including the assembly of clathrin into a precisely shaped lattice tightly associated with the plasma membrane, and the coupling of the lattice to a focused band of actin filaments. Collectively, the outcome of the three specific aims will advance an understanding of how dynamic and functional clathrin coated vesicles emerge from the plasma membrane and couple with a tightly focused band of actin filaments. PUBLIC HEALTH RELEVANCE: Collectively, our results will advance our understanding of how dynamic clathrin-coated vesicles emerge from the plasma membrane and interact with the cytoskeleton. Understanding this mechanism of how clathrin and the cytoskeleton are coupled will contribute to our knowledge of the basis of diseases caused by mutations in proteins associated with these cellular components. With an understanding of how these cellular processes occur, the field of membrane traffic will be able to appreciate how clathrin vesicles form, opening therapeutic strategies for targeting a subset of interactions to, for example, block the entry of opportunistic pathogens that highjack clathrin vesicles for entry, or, conversely, to promote the entry of drugs and receptors that bring cholesterol or insulin into cells via clathrin coated vesicles.